Fig 1.
Reduced sulphation in chronic mouse helminth infection.
(A) HID-AB staining of the caecum of mice with acute infection (BALB/c high dose (HD)) and mice susceptible to chronic infection (AKR HD and BALB/c low dose (LD)) were used to differentiate between sulphomucins (black staining) and sialomucins (blue staining) during T. muris infection. Helminths are highlighted by black arrows. Representative images from n = 5–6 mice per group. Scale bar = 100 μm. (B) Enumeration of the percentage of crypts containing sialomucins during the course of infection. Crypts from BALB/c mice with HD infection were occupied by sulphated mucins at all times. (C) qRT-PCR was used to determine the mRNA levels of Muc2 in BALB/c mice with high and low dose infection and AKR mice with high dose infection. Results from n = 5–6 mice, box plots show median, quartiles and, range. *** = P<0.001. One-way ANOVA with Bonferroni post-test.
Fig 2.
Sulphotransferases are upregulated in epithelial cells following acute infection.
Caecal epithelial cells were isolated and qRT-PCR was used to determine the levels of (A) sulphotransfereases Gal3St1, Gal3St2 and Gal3St3; (B) Sialyltransferases, ST3Gal1, ST3Gal2, ST6GalNAc and ST8GalNac, (C) glucosamine-O-sulphotransferases GlcNAc6ST1, GlcNAc6ST2, GlcNAc6ST3 and GlcNAc6ST4; during acute and chronic T. muris infection. Red dashed line = naïve levels. Results are presented as mean ± SEM of 5–7 mice per group. Unpaired student t-test, *P<0.05, **P<0.01, ***P<0.001, Acute (BALB/c HD) Vs. Chronic (BALB/c LD) infection.
Fig 3.
Changes in mucin glycosylation are dependent on the immune response and are reversed after worm clearance.
(A) Schematic depicting the time course of infection in BALB/c mice in the low dose/high dose reinfection experiment. (B) HID-AB staining depicting the changes in sulpho- and sialomucins during the course of the infection experiment in (A). (C) Worm burdens assessed on day 12, 21, 35, 47, 53 and 56 post infection (pi); red arrow depicts the time of re-infection with a high dose of T. muris eggs. (D) Goblet cell numbers quantified in wild-type, IL-4 knockout (KO) and IL-4R KO uninfected mice and 18 and 32 days after infection with 150 T. muris eggs. (E, F) Caecal tissue from infected and non-infected IL-4 KO and IL-4R KO mice stained with HID-AB; (E) depicts the HID staining intensity measured per 250 goblet cells. Scale bar = 100 μm. Results are presented as mean ± SEM of 5–7 mice per group. One-way ANOVA with Bonferroni post-test. *P<0.05, **P<0.01, ***P<0.001 compared to WT and #P<0.05, ##P<0.01, ###P<0.001 compared to naïve controls.
Fig 4.
Epithelial sulphate transporter Sat1 promotes establishment and is important for the clearance of T. muris infection.
Levels of Sat1 protein and mRNA were determined using (A) immunohistochemistry and (B) qRT-PCR, respectively, in WT and NaS1 KO mice during infection. (C) HID-AB staining illustrates the reduced mucin sulphation in naive Sat1 KO mice compared to WT mice. (D-E,G) Sat1 KO and WT mice were infected with 150 T. muris eggs. (D) Worm burden assessed on day 7 pi.; n = 8 mice per group. (E) Worm burden assessed on day 35, 42 and 56 pi. to confirm chronic infection. (F) Hatching of T. muris eggs determined after 24 h incubation at 37°C with mouse caecum from WT, Sat1 heterozygous mice and Sat1 KO mice under aerobic and anaerobic conditions. (G) ELISA was used to determine cytokine protein levels of IFNγ and IL-13 secreted by T. muris ESP–stimulated leukocytes isolated from mesenteric lymph nodes on day 7 pi. n = 5–6 mice per group. (H) Live worms were treated with RPMI medium only, or with caecal mucus isolated from uninfected WT, NaS1 KO or Sat1 KO mice (n = 12) for 24 h before measuring ATP levels. ATP levels are presented as relative light units per worm. Results are representative of the mean value of 100 worms per group ± SEM. One-way ANOVA with Bonferroni post-test. *P<0.05, **P<0.01, ***P<0.001 compared to WT mice. Scale bar = 100 μm.
Fig 5.
Schematic illustrating the important role of sulphate transporters during helminth infection.
(A) In normal caecal epithelia, NaS1 expressed on the apical surface and Sat1 expressed on the basolateral surface is involved in the uptake of sulphates to maintain sulphate homeostasis and ensure sufficient substrate for sulphation of caecal mucins. (B) During acute T. muris infection, the demand for sulphation increases as a result of goblet cell hyperplasia, leading to the upregulation of NaS1 and Sat1, allowing uptake of sulphates from the luminal surface as well as blood, respectively. (C) In the absence of NaS1 blood sulphate decreases due to decreased intestinal absorption and renal reabsorption. Consequently, Sat1 on the basolateral membrane in the caecal epithelia is upregulated earlier in infection to ensure sufficient uptake of sulphates from blood. This compensation for the deficiency in blood sulphate leads to the recovery of sulphation on mucins ensuring effective worm expulsion. (D) In the absence of Sat1, mice develop hyposulfataemia and the cellular requirement for sulphates is unable to be met by NaS1, leading to reduced mucin sulphation. Reduced mucin sulphation reduces the efficiency of establishment of T. muris infection, but then prevents eradication of the infection.